Experimental and computational investigations of RNA duplexes containing N7-regioisomers of adenosine and LNA-adenosine

RNA has a broad range of roles in cellular processes, including regulation of gene expression, translation, and formation of molecular machinery. Its implication in disease progression makes RNA a precious target for treating currently untreatable disorders. Modified RNA residues offer unique properties that can be utilized to control binding affinity, selectivity, and biostability of RNA. In this study, we conducted comprehensive experimental and computational investigations to elucidate the structural and thermodynamic properties of N7-ribofuranosyladenine (7A) and its locked nucleic acid analog (7AL). Our results demonstrate that 7A and 7AL enhance thermodynamic stabilities of 1×1 mismatches when paired with purines, while exhibiting the opposite effect when paired with pyrimidines. Utilizing NMR and computational techniques, we discovered that 1×1 7A:A and 7AL:A prefer anti-anti conformations, while 1×1 7A:G and 7AL:G prefer syn-anti orientations, both forming two hydrogen bond states, resulting in enhanced duplex stabilities. Additionally, UV melting data indicates that 7A and 7AL enhance duplex stabilities specifically when used at 5′-dangling ends. The unique properties of 7A and 7AL can advance structure-based RNA studies when resolving properties of dynamic RNA regions while stabilizing RNA mismatches involving purines, thereby potentially yielding novel therapeutic strategies targeting RNA-associated diseases.